Project Summary Glioblastoma (GBM) is a primary brain tumor that causes significant neurological deterioration and death. Maximally treated glioblastoma (GBM) recurs in 6 months on average, and it kills most people in less than 2 years. A major reason for inevitable recurrence may be the existence of tumor propagating cells (TPCs), sometimes known as cancer stem cells. No current therapy specifically targets or eradicates TPCs, and our mechanistic understanding of these highly resistant cells is limited. We discovered a transcription factor, ZFHX4, that is required for the TPC state, and its suppression significantly prolongs survival in a mouse xenograft model. The higher the expression of ZFHX4 in patient tumors, the shorter they live. The overall objective of our proposal is to determine why ZFHX4 is required for the oncogenic, TPC state and to determine which aspects of GBM function it controls. We found that ZFHX4 interacts with chromodomain-helicase-DNA- binding protein 4 (CHD4), a core member of the nucleosome remodeling and deacetylase (NuRD) complex. Moreover, we found that, like ZFHX4, CHD4 is required to maintain the TPC state. We also recently discovered that ZFHX4 interacts with SMARCA3 and PARP, which are both required for efficient DNA repair, a hallmark of TPCs. We hypothesize that ZFHX4 transcriptionally drives expression of key GBM genes involved in self-renewal, growth, and migration, and that through interactions with distinct proteins, it also plays a role in DNA damage response. In Aim 1, we will determine the essential function and targets of ZFHX4 in GBM. In Aim 2, we will evaluate how ZFHX4 interacts with CHD4, and determine the biological consequences of their interaction. In Aim 3, we will determine why ZFHX4 is required for TPC survival after DNA damage. We will use biochemical, genetic, and cell biological approaches, including innovative new technologies like calling cards to track ZFHX4 binding sites under specific conditions, super-resolution confocal microscopy to study ZFHX4 localization and inform structure-function analyses, and DNA damaging laser microbeams to study the role of ZFHX4 in DNA damage. Successful execution of these aims will provide important insight into why ZFHX4 is required for the TPC state and fundamental knowledge about this transcription factor and epigenetic regulation in glioblastoma. These studies will help clarify why therapy resistant TPCs cause a significant burden of neurological disease. Our findings will have a significant impact because they will eventually lead to treatments that inhibit ZFHX4 function, reduce the TPC burden in patients, and extend survival.